1. Field of the Invention
This invention relates to macromolecule blotting and more particularly to nucleic acid and protein blotting using a charge modified microporous membrane.
2. Prior Art
Smithies, Zone Electrophoresis in Starch Gels: Group Variations in the Serum Proteins of Normal Human Adults, Biochem. J. 61: 629-641 (1955), showed that starch gel could serve as a molecular sieve through which zone electrophoresis of proteins occur. Since then, there have been constant innovations in the technique of gel electrophoresis. The introduction of acrylamide gels, discontinuous buffer systems, the use of sodium dodecyl sulfate (SDS) to disaggregate protein complexes to be resolved on gels, and the eventual combined use of SDS in discontinuous buffer systems for polyacrylamide gel electrophoresis have been major contributions to the development of one of the most widely used analytical and preparative tools of modern biology.
The main objective of these techniques has been to visually demonstrate the homogeneity or complexity of a protein preparation by following the appearance or disappearance of a particular "band" throughout a given experimental procedure. One-dimensional gels were found to be adequate, provided only relatively simple protein samples such as viruses, bacteriophages, erythrocyte ghost membranes, etc., were being analyzed. More complex systems demanded greater resolving power and new two-dimensional gel systems were developed. Today, even the thousands of polypeptides which are a part of the more intricate proteinaceous samples can be efficiently resolved.
The task of unequivocally correlating a "band" or "spot" with a recognized function has often been difficult, and this is even more so when the resolution of the proteins depends on their denaturization. Nevertheless, many approaches have been developed which allow the identification of a specific enzyme or antigen or glycoprotein or hormone receptor, etc. in a gel. These techniques rely on the ability to maintain at least one of the following prerequisites: (1) that the polypeptides retain their activity throughout electrophoresis; (2) renaturation of a denatured polypeptide; and (3) covalent crosslinkage of the protein in question to a detectable ligand during electrophoresis. Moreover, the actual processing of the gels entails multiple manipulations and extensive incubations and washing procedures. This is very time consuming and quite often prone to handling accidents such as breakage and tearing of wet gels or cracking during the drying of the gels.
In order to try to overcome some of the problems encountered in analyzing gels, a new approach has evolved. A number of reports have been published demonstrating that the well established approach of "Southern-blotting", i.e. transferring DNA patterns from agarose gels to nitrocellulose membranes, can be applied to protein patterns in polyacrylamide gels. Intact protein patterns are eluted from the gels and are immobilized on a substratum. The substratum is, in turn, subjected to the same type of procedures which have been used on gels for "band" or "spot" identification. However, by transferring electrophoretograms to immobilizing matrices one may benefit from the following advantages: (1) wet immobilizing matrices are pliable and easy to handle; (2) the immobilized proteins are readily and equally accessible to various ligands (since the limitations introduced in gels by differential porosity are obviated); (3) transfer analysis generally calls for small amounts of reagents; (4) processing times (incubations and washings) are significantly reduced; (5) multiple replicas of the gels may be made; (6) transferred patterns may be stored for months prior to their use; (7) protein transfers may undergo multiple analyses. Moreover, the transferred protein patterns are amenable to analyses which would be otherwise extremely difficult or impossible to perform on gels.
The term "blotting" today refers to the process of transferring biological macromolecules such as nucleic acids and proteins from gels to an immobilizing matrix. The term is often used in conjunction with the relevant macromolecule, e.g. protein blotting, DNA blotting and RNA blotting. The resulting matrix containing the transferred immobilized macromolecule is known as a "blot" or "transfer" and can be incubated with a ligand, a procedure which may be referred to as "overlay". Thus, for example, immunooverlay, lectin overlay or calmodulin overlay refers to the incubation of a blot with an antibody, lectin or calmodulin, respectively.
DNA blotting, a type of nucleic acid blotting, traces its origin to the technique often referred to as a "Southern Transfer" which was developed by Southern, Detection of Specific Sequences among DNA Fragments Separated by Gel Electrophoresis, J. Mol. Biol. 98: 503-517 (1975). After the chromatographic separation of the DNA fragments, the DNA is denatured while in the gel and the gel is neutralized. The gel is placed between wicking paper which is in contact with a buffer reservoir, nitrocellulose is placed on top of the gel and dry blotting papers are placed on top of the nitrocellulose. Mass flow of buffer through the gel elutes the DNA which then binds to the nitrocellulose. Thus, the electrophoretically separated DNA fragment pattern is transferred and preserved on the nitrocellulose. Hybridization with a specific labeled nucleic acid allows detection of the specific complementary fragments bound to the nitrocellulose.
In 1976, it was discovered that single stranded RNA and DNA could be covalently coupled to a cellulose powder substituted with aminobenzyloxymethyl groups which were activated by diazotizing the amine forming diazobenzyloxymethyl (DBM)-cellulose. This filled a gap in hyberdization technology since RNA does not bind well to nitrocellulose making a Southern Transfer difficult or impossible. In 1977, Alwine, et al., "Method for Detection of Specific RNAs in Agarose Gels by Transfer to Diazobenzyloxymethyl-Paper and Hybridization with DNA Probes, Proc. Natl. Acad. Sci. U.S.A., 74: 5350-5354, prepared a cellulosic fibrous sheet (i.e. blotting paper) derivatized with diazobenzyloxymethyl groups, termed DBM-paper, viz. ##STR1## which could be used for transfer of an electrophoretically separated pattern of RNA from an agarose gel in a method similar to a Southern Transfer. Aminophenylthioether paper activated to the diazo form (DPT-paper) has also been used. Both papers covalently and irreversibly couple DNA, RNA and proteins.
DBM-paper and DPT-paper having the disadvantages that they require activation, have limited life, i.e. their activity is labile, having binding capacity only comparable to nitrocellulose, irreversibly couples the macromolecules, thus preventing their subsequent elution, and may present difficulties in resolution due to the texture of the surface.
In an effort to overcome some of the inconveniences of DBM paper, Thomas, (1980) developed a technique to transfer RNA and small DNA fragments to nitrocellulose using high salt concentrations. The binding efficiency of RNA was found to be 80 ug/cm.sup.2 as compared to 35 ug/cm.sup.2 for DBM paper. Elution of macromolecules from polyacrylamide gels can be accomplished efficiently by electrophoresis. However, the requirement of high salt concentrations would lead to impractically high currents.
Although first developed in connection with the study of DNA and RNA, it became recognized that the blotting techniques pioneered by Southern could be applied to proteins. The protein transfer techniques were first developed by Renart, Transfer of Proteins from Gels to Diazobenzyloxymethyl-Paper and Detection with Antisera: A Method for Studying Antibody Specificity and Antigen Structure, Proc. Natl. Acad. Sci. U.S.A. 76: 3116-3120 (1979), who achieved protein transfer to DBM paper using composite agarose-acrylamide gels in which the acrylamide crosslinking agent was reversible. After electrophoresis, the crosslinking was removed, leaving a low percentage agarose gel from which the proteins transferred easily. Shortly thereafter, a protein blotting procedure was developed by Bowen et al., The Detection of DNA-Binding Proteins by Protein Blotting, Nuc. Acids Res. 8: 1-20 (1980), using nitrocellulose for the transfer of DNA binding and other ligand-binding proteins separated on SDS-polyacrylamide gels. In this procedure, transfer was accomplished by diffusion. Towbin, et al. (1979) Proc. Natl. Acad. Sci. USA 76: 4350-4354 and Bittner, M. et al. (1980) Anal. Biochem. 102: 459-471 had demonstrated that transfer could be accomplished electrophoretically even at low salt concentrations.
In general, protein blotting should be viewed as two sequential events, namely the elution of the polypeptide from the gel and the adsorption of the eluted material to an immobilizing matrix.
Three main driving forces have been exploited for macromolecule elution. One is diffusion. Here, the gel containing the macromolecules to be transferred is sandwiched between two sheets of immobilizing matrix which are in turn sandwiched between foam pads and stainless steel screens. This final assembly is then submerged in two liters of buffer and allowed to sit for 36-48 hours. The result of this incubation is that two identical replica blots are obtained. The efficiency of transfer may reach 75%, but this value must be divided between the two replicas. If the amount of macromolecule adsorbed onto the matrix is sufficient for the intended assays and the long transfer time is not detrimental, blotting by diffusion can be useful.
The second means of macromolecule blotting is essentially based on mass flow of liquid through the gel in the same manner DNA blots are achieved in the traditional procedure described by Southern. The gel is placed in a reservoir of buffer. A matrix is applied to the gel and paper towels are piled onto the matrix. The towels adsorb the buffer from the reservoir through the gel and matrix. This movement of fluid serves as a driving force which elutes the proteins out of the gel which are then trapped in the filter. This technique is less time consuming than diffusion blotting and the efficiency of elution is better. A modification of this approach has been suggested which allows bidirectional blotting. Moreover, the time for efficient elution has been dramatically reduced by applying a vacuum to facilitate the process.
The most widely used mode for protein blotting is based on electroeluting the proteins from gels. This is made possible due to the fact that proteins, in contrast to DNA, adsorb the nitrocellulose even in low ionic strength buffers (when other immobilizing matrices are used, e.g. DBM paper, this is no longer a consideration). Therefore, one can electrophorese the proteins out of the gel without generating intolerable currents. It should be noted that the concept of electroelution of macromolecules for blotting was originally described by Arnheim and Southern in 1977 (Heterogeneity of the Ribosomal Genes in Mice and Men, Cell 11: 363-370). Numerous apparatus designs have been reported and quite a few are now commercially available. In essence, a wet matrix material is placed on a gel, making sure that no air bubbles are caught within the filter or between the matrix and the gel. The matrix and gel are then sandwiched between supportive porous pads such as "Scotch Brite" scouring pads, foam rubber or layers of wet blotting paper. The assembly is then supported by solid grids (usually nonconductive). It is very important that the gel and matrix are firmly held together. This ensures good transfer and prevents distortion of the protein bands. The supported "gel+matrix sandwich" is inserted into a tank containing "transfer buffer" and placed between two electrodes. The electrodes, which may be tacked to the sides of the tank, are designed so as to generate a homogeneous field over the entire area of the gel which is to be transferred. Continuous conductive sheets can serve as electrodes and theoretically are most appropriate for this purpose. Slabs of graphite and stainless steel plates have been used. However, operating units with such electrodes is usually impractical due to the requirement for excessively high currents. Some apparatus use stainless steel mesh or platinum mesh. An economical, yet efficient, design that seems to work reasonably well is that described by Bittner et al (Electrophoretic Transfer of Proteins and Nucleic Acids from Slab Gels to Diazobenzyloxymethyl Cellulose or Nitrocellulose Sheets, Ana. Biochem. 102: 459-471). Factors that may influence the homogeneity of the field are the distance between the gel and the electrode and the density of the electrode material i.e. the distance between each stretch of wire used. In general, the more electrode material present the lower the electrical resistance of the system. This, therefore, demands higher currents in order to obtain reasonable voltage differences to drive the elution process. The transfer buffers can be of low ionic strength, such as a phosphate buffer, Tris-borate buffer or Tris-glycine and may or may not contain methanol. Methanol tends to increase the binding capacity of nitrocellulose for protein and stabilize the geometry of the gel being transferred but reduces the elution efficiency of protein from SDS gels and in its presence, electroelution must be carried out for long durations, generally more than 12 hours, in order to obtain efficient transfer of high molecular weight proteins. In the absence of methanol, gels of acrylamide tend to swell and, if allowed to occur during protein transfer, causes the band to be distorted.
The conditions of transferring per se are dependent on the type of gel, the immobilizing matrix and the transfer apparatus used as well as the macromolecules themselves. Nondenaturing gels, SDS gels, lithium dodecyl sulfate containing gels, iso-electrofocusing 2D gels and agarose gels have all been used for protein blotting. It is necessary to determine the electric charge of the protein to be eluted and place the matrix on the appropriate side of the gel. Proteins from SDS-gels, for example, are eluted as anions and therefore the matrix should be placed on the anode side of the gel. The case may be the opposite for nondenaturing gels. As electroelution progresses, electrolytes from the gel are also eluted and contribute to the conductivity of the buffer, resulting in a drop in resistance. Were one to conduct the transfer at constant voltage, the current would increase accordingly and currents in excess of one ampere, and indeed up to five amperes, may develop. High current (one to five ampere) power supplies are now commercially available for electroblotting. Another alternative has been to use a 12 V battery charger which seems to be quite adequate. However, as most of the common paper supplies used for gel electrophoresis cannot exceed 200-250 mA, it has been found advantageous to run at a maximal constant current (i.e. 200 mA) and allow the voltage to gradually drop during the transfer.
There are incidences where isolated proteins of low or moderate molecular weight do not elute efficiently from a gel. This can occur in those cases where these proteins fortuitously are at their isoelectric point and have no tendency to migrate in the electric field exerted. In such an instance, other buffer conditions can be employed.
There are numerous immobilizing matrices which are available today. While nitrocellulose is the most widely used material as a matrix, the interaction of a protein with the nitrocellulose is complex and not clearly understood. For example, at pH 8 where protein electroelution is usually performed, nitrocellulose is negatively charged as are the proteins being adsorbed. Hydrophobic effects play a role in this interaction and indeed, protein elution from the matrix is facilitated by non-ionic detergents. Some proteins, especially those of low molecular weight, bind with low affinities to nitrocellulose and may be lost during transfer or subsequent processing. To prevent such a loss, the transferred polypeptide can be crosslinked to the matrix, thereby covalently stabilizing the protein pattern. The presence of cellulose acetate in nitrocellulose membrane matrices seems to reduce their capacity to bind protein. However, cellulose acetate matrices have been employed successfully for protein blotting.
In order to remedy some of the disadvantages of nitrocellulose matrices, alternative immobilizing matrices have been proposed. Transfer to DBM paper results in a covalently bound and stable protein pattern. Resolution is slightly lower on this material due to its intrinsic coarseness as compared to membrane matrices. Also, glycine, a commonly used material in transfer buffers, can interfere with DBM paper protein blotting. There still remains, therefore, a need for an immobilizing matrix which overcomes the disadvantages of the nitrocellulose, DBM paper and other matrices which have been used heretofore. A greater transfer capacity is also desirable.
It is known in the art that membranes may be used in chromatography in general and electrophoresis in particular. See, for example, U.S. Pat. Nos.:
3,808,118 to Golias PA1 3,829,370 to Bourat PA1 3,945,926 to Kesting PA1 3,957,651 to Kesting PA1 3,989,613 to Gritzner PA1 4,043,895 to Gritzner PA1 4,111,784 to Dahms PA1 4,158,683 to Del Campo PA1 4,204,929 to Bier PA1 4,243,507 to Martin PA1 4,310,408 to Roe and PA1 4,311,574 to Ishikawa. PA1 3,418,158 to Perry and PA1 3,523,350 to Goldberg.
Additionally, it is known that polyamide powders may be used to perform chromatographic separations. See, e.g. U.S. Patents Nos.:
Hiratsuka et al., U.S. Patent 4,128,470, teaches that nylon microporous membranes may be used in electrophoresis and isoelectric focusing as the medium through which chromatography is performed.
New England Nuclear has been marketing an uncharged nylon membrane for use in blotting in its "Gene Screen" electrophoresis product line. This material appears to have equivalent binding capacity for proteins as nitrocellulose.
3. Utility
Macromolecule blots or transfers can be "overlaid" with a variety of reagents in much the same manner that has been developed for gels. Manipulation of filter matrices is less time consuming, more economical with respect to the reagents used and is less exposed to handling accidents. More important, however, is the fact that transferring proteins from gels to matrices in effect eliminates diffusion barriers. Furthermore, denatured polypeptides can sometimes be renatured upon removal of SDS from them and this process is probably much more convenient and effective using blots.
Presently, most of the probes which have been used are macromolecules which specifically bind well to defined domains of the polypeptides under investigation. Lectins have allowed the detection of glycoproteins and antibodies and the identification of their corresponding antigens. Regardless of what the intended assay may be, vacant areas of the matrix which do not contain protein bands can non-specifically adsorb probes during the overlay process leading to intolerable background. Therefore, the unbound sites of the matrix must be quenched prior to overlaying the blot. Quenching is most commonly achieved by incubating the blot in high concentrations of bovine serum albumin (BSA) or hemoglobin at 25.degree.-60.degree. C. for 1-12 hours. Other materials such as ovalbumin, gelatin and various animal sera have also been used. The temperature, choice of protein and duration of quench depend on the type of matrix material and the probe being used. Nonionic detergents have also been included in quench or washing buffers to reduce non-specific binding.
Once the blot has been quenched, it is reacted with the probe. In general, all reactions should be carried out in the presence of quenching protein. The reacted blot is then washed extensively in buffer (which does not have to contain protein). If the probe is itself radioactive or conjugated to an enzyme or fluorescent tag, the blot can be immediately autoradiographed, reacted with the relevant substrate or visualized in UV light, respectively. If further second or third reagents are necessary to detect the presence of probe-band complexes, then each consecutive reagent is incubated with the blot followed by a wash. The great sensitivity of these overlay techniques has allowed the detection of very low amounts (e.g. 1 ng) of viral antigens in natural fluids. Furthermore, these techniques have also been employed in the analysis of human sera of patient suffering from various immune disorders.
One of the advantages of blots over gels is that they may be reused or subjected to multiple reactions. Once a signal has been obtained and recorded, the blot may be "erased" by removing the probe but retaining the original protein pattern on the matrix. The "erased" filter can be reused for additional overlay analysis for further characterization of the elements of the gel pattern. "Erasing" can be accomplished by dropping the pH to dissociate antibody-antigen complexes or by denturing the probe by incubating the blot in urea or SDS. Selective dissociation of probe-band complexes, demonstrating specificity, may also be achieved. Lectins can be selectively competed off with relevant haptens. Calmodulin can be dissociated from calmodulin binding proteins by removing Ca++ from the system. These reactions can still be performed even after the protein blot has been autoradiographed to obtain the initial signal.
Use of protein blots usually has the objectives of demonstrating protein-protein or protein-ligand interactions or of exploiting the production of an immobilized polypeptide as an intermediate step in immunological or biochemical analyses. Both of these approaches have been exploited in novel usages of protein blots. For example:
a: Analysis of protein-ligand associations
DNA-protein and RNA-protein interactions have been analyzed by protein blots. Histone H2 associations with H3 and H4 have also been demonstrated.
The epidermal growth factor receptor was identified by hormone overlaying a transferred membrane pattern. Membranes from human epidermoid carcinoma cells (A-431) were prepared and run on SDS-polyacrylamide gels. The gels were then electroblotted onto DBM paper, quenched and overlaid with epidermal growth factor and subsequently with radioactively labeled antibody to the hormone. One very predominant signal at 150 KD was detected.
b: Identification of enzyme subunits
The detection of an inactive enzyme on a protein blot has been demonstrated. Phosphodiesterase I, for example, was boiled in 2% SDS for 5 minutes and run on a SDS-polyacrylamide gel. Protein was blotted into nitrocellulose filters which were reacted with excess antiphosphodiestrase I. The matrix was then incubated with a crude preparation containing active enzyme. The active enzyme bound via unoccupied sites of the antibody to the inactivated resolved subunit immobilized on the matrix. The matrices were then reacted for enzyme activity and the immunocomplexes were thus detected.
c: Affinity purification of monospecific antibodies
Blots have been used for the purification of monospecific antibodies. Polypeptides are resolved on SDS-polyacrylamide gels and blotted onto DBM or CNBr paper. The matrices were overlaid with serum containing polyclonal antibodies. Then single hands containing antigen-antibody complexes were excised from the matrices from which the monospecific antibody was eluted by incubating the strip in low pH (2-3) buffer. The eluted probe could then be used for immunocytochemical localization studies.
d: Demonstration of cell-protein interactions
Protein blots have been used to demonstrate specific whole cell-polypeptide interactions. Human plasma was run on SDS-polyacrylamide gels and transferred to nitrocellulose matrices. Once the matrices were quenched, they were incubated with normal rat kidney cells (NRK cells) which specifically bound to immobilized polypeptides presumably involved in cell attachment. The cells were stained with amino black and were found to locate themselves at two discrete bands. These bands were identified as: fibronectin and a newly discovered 70 KD entity.